Peptide substrates for chymosin (Rennin) Kinetic studies with bovine κ-casein-(103–108)-hexapeptide analogues

Visser, S.; Rooijen, P.J. van; Schattenkerk, Cecile; Kerling, K. E. T.

doi:10.1016/0005-2744(77)90148-6

Cited by 33 publications

(22 citation statements)

References 17 publications

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“…The rate of the enzymatic cleavage of short 103-109 peptide is considerably lower than that of long 98-109 peptide. This result correlates with the data on the cleavage of analogous peptides by chymosin described by Visser et al [10][11][12] who observed a marked decrease in Km for the long peptide, and emphasized a particular role of residues at positions Ps P4 in the enzyme reaction.…”

Section: Resultssupporting

confidence: 91%

“…Therefore, the isolated histid- ine-proline cluster can be considered as the allosteric activator. The comparison of the hydrolysis rates for short 103-109 and long 98-109 peptides, as well as the data obtained by Visser et al [10][11][12], show that the cluster induces the allosteric-like activation of chymosin when incorporated into a substrate. A prominent effect of the histidine-proline cluster on the Km of x-casein fragments [10] shows that the attachment of the cluster promotes the structural changes in the enzyme binding pockets making them more accessible for the substrate.…”

Section: Sepb ~supporting

confidence: 58%

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Post X‐ray crystallographic studies of chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine‐proline cluster of κ‐casein

Gustchina¹,

Румш²,

Ginodman³

et al. 1996

FEBS Letters

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Calf chymosin molecules exist in the two alternative structural forms: the first one has S~ and $3 binding pockets occluded by its own Tyr 77 residue (the self-inhibited form); the second has these pockets free for a substrate binding (the active form). The preliminary incubation of the enzyme with a pentapeptide corresponding to the histidine-proline cluster of the specific substrate it-casein results in a 200-fold increase of the hydrolysis rate for the enzyme 'slow substrate'. The result suggests that the cluster is an allosteric effector that promotes the conversion of the enzyme into the active form. These data provide the experimental ground for the explanation of chymosin specificity towards K-casein.

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Section: Resultssupporting

confidence: 91%

Section: Sepb ~supporting

confidence: 58%

Post X‐ray crystallographic studies of chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine‐proline cluster of κ‐casein

Gustchina¹,

Румш²,

Ginodman³

et al. 1996

FEBS Letters

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“…In earlier studies [7,8] a raughly elevenfold and eightfold decrease was found at pH 4.7 for x(103-108) OMe and x(103-112) re-spectively, after partial oxidation of Met-106. In this respect it is interesting to note that Kaye and Jolles [30] conclude from oxidation experiments with whole x casein that the integrity of Met-I06 is not essential for chyrnosin activity.…”

Section: Comparison O J Kinetic Parametersmentioning

confidence: 84%

“…Data on selected synthetic x casein fragments [7] as well as on whole x casein [9,28,29] have been added for comparison. From this it follows that, whereas the presence of Leu-103 is of utmost importance [7,8], a further improvement of the substrate properties (predominantly determined by a lower K,) is obtained by adding the 98-102 fragment His-Pro-His-Pro-His-to the sequence. Tight binding of this part of the substrate molecule onto its enzyme counterpart via exclusion of water molecules may form an explanation for the sharp decrease in K,, if one accepts that K,,, approaches the enzymesubstrate dissociation constant K, [9].…”

Section: Comparison O J Kinetic Parametersmentioning

confidence: 90%

Peptide Substrates for Chymosin (Rennin) Isolation and Substrate Behaviour of Two Tryptic Fragments of Bovine kappa Casein

1980

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From a tryptic digest of bovine 1.1 casein two peptides with the sequences 98-111 and 98-112 have been isolated. Both fragments contain the chymosin-sensitive Phe-105 -Met-106 peptide linkage, the rapid cleavage of which forms the first step of the casein coagulation during the milk-clotting process.At pH 5.3-5.5, where the rate of cleavage of the two peptides by chymosin is maximal, the proteolytic coefficient k,,,/Km of the enzymic reaction is 4-5 pM-' s-I. At p H 6.6 (pH of milk) this parameter is around 2 pM-' s-', which is very close to that found for whole x casein, the natural substrate for chymosin in the milk. By performing the rate experiments at pH 4.7 the kinetic parameters for the splitting of the tryptic peptides could be compared with those obtained earlier [Visser, S., van Rooijen, P. J., Schattenkerk, C., and Kerling, K . E. T. (1976) Bioclzim. Biophyys. Acta, 438, 265 -2721 with shorter, synthetic peptide substrates. From this it appeared that addition of the 98 -102 segment (His-Pro-His-Pro-His-) to the 103-112 sequence brings about a 30-fold improvement of the overall substrate properties, which is predominantly determined by a decrease of K,.The pH dependence of the hydrolysis of the tryptic fragments indicates the apparent pK1 and pK2 values of catalytically important groups on the enzyme-substrate complexes to be in the regions 4.0 -4.2 and 6.5 -6.7 respectively.The rate of cleavage of the x casein (98 -112) pentadecapeptide is diminished by raising the ionic strength of the reaction medium between 0.01 and 0.1, and also by partial oxidation of the Met-106 residue. The latter is in agreement with earlier results obtained with short-chain substrates.

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“…Leu-Ser-PheMet-Ala, that there is any appreciable hydroly~is.~-'~ Extending the N -and C-terminal ends of the pentapeptide results in an increase in this rate of hydrolysis. 6 It has also been shown that a peptide obtained from a L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) tryptic digest of rc-casein, rcCN(98-111) (HisPro-His-Pro-His-Leu-Ser-Phe-Met -Ala -He-Pro -ProLys), contains all the determinants necessary to render the Phe-Met bond as susceptible to hydrolysis by chy-* Author to whom correspondence should be addressed.…”

Section: Introductionmentioning

confidence: 97%

Proton assignment and structural features of a peptide from the chymosin‐sensitive region of bovine k‐casein determined by 2D‐NMR spectroscopy

Plowman

Smith

Creamer

et al. 1994

Magnetic Reson in Chemistry

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The peptide ~-casein(98-111) corresponding to the sequence 98-1 11 of bovine Kcasein (His-Pro-His-Pro-His-LeuSer-Phe-Met-Ala-Ile-Pro-Pro-Lys), which includes the aspartic protease sensitive region, was analysed by 2D 'H NMR spectrascopic techniques in DMSO-d, . The spectral peaks of all protons resolved at 400 MHz were assigw ed. The changes in the amide proton shifts with pH and temperature and the existence of particular crow peaks in the ROESY spectra were consistent with the central region of the peptide being in an extended chain in solution but with some type of distinct conformations at the N-and C-termini. The results are not consistent with the peptide having any helical structure at either low or high pH.

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Peptide substrates for chymosin (Rennin) Kinetic studies with bovine κ-casein-(103–108)-hexapeptide analogues

Cited by 33 publications

References 17 publications

Post X‐ray crystallographic studies of chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine‐proline cluster of κ‐casein

Post X‐ray crystallographic studies of chymosin: the existence of two structural forms and the regulation of activity by the interaction with the histidine‐proline cluster of κ‐casein

Peptide Substrates for Chymosin (Rennin) Isolation and Substrate Behaviour of Two Tryptic Fragments of Bovine kappa Casein

Proton assignment and structural features of a peptide from the chymosin‐sensitive region of bovine k‐casein determined by 2D‐NMR spectroscopy

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